The present invention relates to oral vaccines, particularly those provided by edible plants. The invention employs genetic engineering techniques to produce transgenic plants capable of expressing immunogenic polypeptides, including hepatitis B antigen (HBsAg) in quantities sufficient to elicit an immune response in a human or animal that consumes all or a part of the plant.
A vaccine for hepatitis B was the first xe2x80x9cnew generationxe2x80x9d recombinant vaccine licensed by the FDA for human use. The immunogenic subunit in this formulation is produced by expressing the gene encoding HBsAg in recombinant yeast; the protein is purified from the genetically engineered yeast and is used for parenteral delivery. In the developed world, the recombinant vaccine has displaced the use of an earlier vaccine derived from the plasma of infected individuals. Both plasma-derived and rHBsAg vaccines are shown to be reasonably safe and effective in high-risk adult populations and newborn infants. However, the cost of the rHBsAg vaccine prevents its extensive availability in developing countries.
The envelope of the hepatitis B virus (HBV) contains three size classes of proteins that share carboxy-terminal sequences. These proteins, called hepatitis B surface antigens (HBsAgs), include large (L, containing a pre-S2 domain), medium (M1 containing a pre-S1 domain), and small (S, containing only the S domain) size classes. All three proteins are found in infectious virions (often referred to as Dane particles) recovered as 42 nm spheres from the serum of infected patients. Serum samples also contain empty spherical particles averaging 22 nm, which contain primarily S class proteins. Mammalian cell lines transfected exclusively with DNA encoding the S protein release 20 nm empty spheres similar to those from infected cells. Moreover, yeast cells transformed with the same gene form analogous spheres, which are found to be equally immunogenic as the 22 nm spheres from infected cells. The yeast-derived material forms the active constituents of the currently available commercial vaccines ENGERIX (SKB) and RECOMBIVAX (Merck).
In mammalian cells, newly synthesized L, M, or S proteins insert into the membrane of the endoplasmic reticulum (ER). The S protein consists of 226 amino acids; its N and C-termini are thought to be on the ER lumen side and it has four transmembrane helices. All three proteins have a glycosylation site at position 146 of the S domain. The three proteins differ in the available sites for and the extent of glycosylation. Improper glycosylation can prevent virion formation, presumably due to misfolding of the proteins. Also, multiple disulfide bonds among cysteines in the proteins are known to be important to the structure of the assembled virion or particles and to the structure of antigenic loops in the protein. Incorrectly assembled particles may interact with cellular chaperones because of the incorrect folding to prevent secretion.
Higher levels of preS1 and preS2 are present in HBV than in 17-25 nm HBsAg particles therefore, immunization with HBsAg particles may not generate high titer antibodies to the preS sequences expressed on HBV. During the course of HBV infection in humans the levels of preS proteins increase during active replication, and anti-preS antibodies and T cells are generated prior to S protein-specific responses. Once anti-HBsAg antibodies rise, anti-preS antibodies decline.
The roles of preS1 and preS2 in virus attachment and neutralization have led to the development of vaccines containing these sequences as well as the entire S region. Vaccines incorporating preS sequences include HEPAGEN (Merck) and BIO-HEP-B (BTG); both are produced in mammalian cell lines. In formulating whole particles that contain S and preS proteins it is important to note that the relative amounts of S, M, and L proteins affect HBsAg assembly, e.g., high levels of L protein reduce the amount of HBsAg particle formation and secretion.
The assembly of the S, M and L surface proteins into particles occurs during budding of the complex into the ER, followed by transport of the particles through the Golgi apparatus to the exterior of the cells. Nanometer scale biological structures, such as viral capsids, assemble through polymerization of similarly folded protein subunits using a small number of well-defined bonding contacts. The driving force for polymerization is the formation of favorable bonding interactions as free subunits are incorporated into the growing polymer. For envelope proteins such as HBsAg, essential steps in the polymerization process are appropriate integration of the polypeptide into the ER membrane followed by establishment of contact among the protein subunits. Normal cellular transport and sorting of proteins in the endomembrane system may contribute to this process.
U.S. Pat. No. 4,710,463 to Murray proposes a method of producing a polypeptide having the antigenicity of a hepatitis B core or surface antigen, which employs a unicellular host. U.S. Pat. No. 5,738,855 to Szu et al. proposes a modified oligosaccharide immunogen similar to the Vi antigen of Salmonella typhi, which can be conjugated to a carrier, such as hepatitis B surface antigen. U.S. Pat. No. 4,847,080, EU 0154902 B1, and subsequent papers of Neurath et al. identify peptide epitopes in the preS1 and preS2 regions for both hepatocyte binding and neutralization, as well as peptides that can be included in a vaccine.
Mammals infected by a pathogen mount an immune response when overcoming the invading microorganism by initiating at least one of three branches of the immune system: mucosal, humoral, or cellular immunity. Mucosal immunity largely results from the production of secretory IgA antibodies in secretions that bathe mucosal surfaces in the respiratory tract, the gastrointestinal tract, the genitourinary tract, and the secretory glands. These mucosal antibodies act to limit colonization of the pathogen on mucosal surfaces, thus establishing a first line of defense against invasion. The production of mucosal antibodies can be initiated by local immunization of the secretory gland or tissue or by presentation of the antigen to either the gut-associated lymphoid tissues (GALT; Peyer""s Patches) or the bronchial-associated lymphoid tissue (BALT).
Mucosal immunization can be achieved by oral presentation of antigens. Specialized epithelial cells (M cells) overlying organized mucosal lymphoid tissues along the intestinal tract sample the antigens by taking up (by endocytosis) infectious bacteria, viruses, and macromolecules. These are passed to the underlying follicles where immune responses are initiated and cells are dispersed to both mucosal and systemic immune compartments. Epithelial cells are also an integral component of the regulatory cytokine network, including those that are important in the differentiation of B cells.
Oral immunization also induces strong humoral immune responses. Humoral immunity results from the production of circulating antibodies in the serum (especially IgG and IgM), precipitating phagocytosis of invading pathogens, neutralization of viruses, or complement-mediated cytotoxicity against the pathogen. A well-documented relationship exists between HBV protection and the amplitude of the systemic antibody and T cell response to HBsAg proteins, and this protection is likely to be achieved by oral immunization.
In contrast to the large variety of currently available injectable vaccines that provide systemic immunity, vaccines administered non-systemically to stimulate mucosal immunity are rare. Recently, however, there has been a surge of interest in developing novel strategies for vaccine development with oral delivery as the preferred route of delivery. In the design of a successful oral vaccine, two aspects deserve special attentionxe2x80x94the use of an appropriate adjuvant and the development of an appropriate antigen delivery system.
Most protein antigens studied for use as adjuvants when administered orally in large doses fail to provoke a mucosal antibody response, but instead induce a state of unresponsiveness or oral tolerance. Cholera toxin (CT) and E. coli heat-labile toxin (LT) are exceptions. The feeding of either CT or LT does not induce tolerance for antibody response and additionally can prevent induction of oral tolerance to unrelated antigens that are administered orally along with the CT or LT (Elson, C. et al., J. Imnmunol. 133:2892-2887 (1984)). These results suggest that CT/LT direct the overall outcome in favor of responsiveness rather than tolerance. In other studies, it is found that CT does not increase the immune response against an antigen that has been previously fed without CT (Xu-Amano, J. Exp. Med. 178:1309-1320 (1993)). This is significant because it indicates that no immune response is mounted against normal dietary antigens in the presence of CT. Generally, two different approaches are taken to induction of tolerance versus immunization following the oral administration of antigen: for the induction of oral tolerance, soluble or aqueous antigen is administered alone; whereas for vaccination protocols, antigens are usually administered in conjunction with mucosal adjuvants.
Recent advances in genetic engineering have provided the tools necessary to transform plants as relatively low-cost candidates for the expression of immunogenic proteins. Both monocotyledonous and dicotyledonous plants have been stably transformed. For instance, tobacco, a dicot, has been transformed with a gene encoding the S protein and the cells can be disrupted to release spheres (or virus-like particles; VLPs) (Mason et al., PNAS USA, 89: 11745-11749 (1992)). When injected into mice, the particles qualitatively mimic the immunogenic properties of the commercial vaccine (Thanavala et al., PNAS USA, 92: 3358-3361 (1995)). This suggests that the process of protein synthesis and assembly may be similar in plant and mammalian cells. Unfortunately, low rates of protein synthesis in the transgenic plants have precluded detailed studies of the formation of the VLPs in vivo.
U.S. Pat. No. 5,679,880 to Curtiss, III proposes a transgenic plant that expresses an antigen or antigenic determinant of a pathogenic microorganism that may elicit a secretory immune response in an animal. U.S. Pat. Nos. 5,484,719 and 5,612,487 to Lam et al. disclose obtaining antigenic particles from transgenic tobacco transformed to express HBsAg. Presently, no method exists to predict the efficiency by which newly synthesized HBsAg proteins will polymerize into antigenic structures in plant cells since the rate-limiting factor(s) are not known. Accordingly, previous studies showing particle formation in transgenic plants may involve non-specific insertion of the newly synthesized protein into various cellular membranes, which could explain the low level of particles accumulating in transgenic plants described to date.
It is desired to develop an inexpensive, safe, and highly effective oral vaccine that elicits systemic, and preferably, mucosal immunity to prevent hepatitis B virus infection. Such a vaccine should be based upon a hepatitis B antigen or primary epitope thereof. An approach that could increase formation of HBsAg virus like particles can employ fusion proteins designed to alter the cellular targeting of the newly synthesized protein subunits. The fusion proteins can have, for instance, a N-terminal leader peptide known to enter the endomembrane system or a C-terminal KDEL (SEQ ID NO:22) extension known to regulate microsomal retention. A particularly satisfactory approach for application in developing countries would be the development of transgenic plants having edible parts that consistently establish oral immunity against HBV when consumed.
It is an object of the invention to provide plant expression vectors comprising at least two expression cassettes that function to reduce transcriptional silencing of polynucleotide expression. It is another object of the invention to provide novel plant expression vectors for expression of immunogenic polypeptides, including HBsAg. The plant expression vectors can be used to produce immunogenic polypeptides, including HBsAg, in edible plant tissues. It is a further object of the invention to provide such immunogenic polypeptides in edible plant tissue to elicit an immune response in humans and animals when the plant tissues are consumed. These and other objects of the invention are provided by one or more of the embodiments described below.
One embodiment of the invention provides a plant expression vector comprising two expression cassettes. The first cassette comprises a polynucleotide encoding an antigen and the second expression cassette comprises a polynucleotide encoding the same antigen as the polynucleotide of the first expression cassette. The polynucleotide of the second expression cassette is non-identical to the polynucleotide of the first expression cassette. The first cassette can optionally comprise a polynucleotide encoding a hepatitis B surface antigen (HBsAg) and the second expression cassette can comprise a non-identical polynucleotide encoding a HBsAg. Optionally the first expression cassette can comprise a polynucleotide encoding a plant-optimized HBsAg polypeptide, preferably, with at least one plant optimized codon, and wherein the second expression cassette comprises a polynucleotide encoding a native virus-derived HBsAg polypeptide. Even more preferably, the polynucleotide of the first expression cassette comprises SEQ ID NO:3 and the polynucleotide of the second expression cassette comprises SEQ ID NO:1.
Preferably, gene silencing, including RNA-mediated transcriptional gene silencing, is reduced or eliminated when both polynucleotides are expressed in a cell. Optionally, the plant expression vector can have a polynucleotide of the first cassette and a polynucleotide of the second cassette wherein said polynucleotides comprise no more than 90 or no more than 60 contiguous identical nucleotides.
The plant expression vector may further comprise a first expression cassette farther comprising a 5xe2x80x2 transcribed, untranslated region and a second expression cassette comprising a non-identical 5xe2x80x2 transcribed, untranslated region. The plant expression vector can also comprise a first expression cassette further comprising a 3xe2x80x2 transcribed, untranslated region and a second expression cassette comprising a non-identical 3xe2x80x2 transcribed, untranslated region. Additionally, the plant expression vector can comprise a first expression cassette further comprising a 5xe2x80x2 transcribed, untranslated region and a 3xe2x80x2 transcribed, untranslated region. The second expression cassette can comprise a non-identical 5xe2x80x2 transcribed, untranslated region and a non-identical 3xe2x80x2 transcribed, untranslated region as compared to the first expression cassette. The first expression cassette can comprise a TEV 5xe2x80x2 transcribed, untranslated region and a vspB 3xe2x80x2 transcribed, untranslated region, and a second expression cassette comprising a TMV 5xe2x80x2 transcribed, untranslated region and apin2 3xe2x80x2 transcribed, untranslated region. The plant expression vector can additionally comprise a first expression cassette comprising a plant-optimized HBsAg polypeptide and a second expression cassette comprising a native virus-derived HBsAg polypeptide.
In still another embodiment of the invention, an E. coli cell is transformed with the plant expression vector described above. Virus like particles can assemble in the cell.
In yet another embodiment of the invention, an Agrobacterium cell is transformed with the plant expression vector described above. Virus like particles can assemble in the cell.
In another embodiment of the invention a plant cell is transformed with the plant expression vector described above. Virus like particles can assemble in the cell. Preferably the plant cell is selected from the group consisting of tomato, potato, banana, and carrot cells. Even more preferably, the plant expression vector is integrated into the nuclear genome of the plant cell.
In even another embodiment of the invention a plant seed comprising the plant expression vector described above is provided.
In still another embodiment of the invention a polynucleotide comprising a nucleic acid sequence encoding a hepatitis B surface antigen (HBsAg) is provided. The polynucleotide is operably linked to a plant functional promoter; a translation enhancement sequence; and a termination sequence. The polynucleotide lacks an untranscribed region between the translation enhancement sequence and the HBsAg encoding sequence. The nucleic acid sequence encoding the HBsAg can comprise at least one altered codon, wherein the altered codon is a plant preferred codon. The plant-functional promoter can be selected from the group consisting of cauliflower mosaic virus (CaMV) 35S, tomato E8, ubiquitin, mannopine synthase, patatin, and granule-bound starch synthase (GBSS) promoters. The promoter may include a dual enhancer region. The translation enhancement sequence can be selected from the group consisting of tobacco etch virus (TEV) and tobacco mosaic virus (TMV) omega translation enhancers. The termination sequence may be selected from the group consisting of a nopaline synthase (nos), a vegetative storage protein (vsp), or a proteinase inhibitorxe2x80x942 (pin2) termination sequence.
Further the polynucleotide may lack an untranscribed region between the HBsAg encoding sequence and the termination sequence. The polynucleotide can further comprise a nucleic acid sequence encoding a microsomal retention signal operably linked to the 3xe2x80x2 end of the HBsAg encoding sequence. The microsomal retention signal can be Ser-Glu-Lys-Asp-Glu-Leu (SEQ ID NO:4). The polynucleotide can lack an untranscribed region between the microsomal retention signal and the termination sequence. The polynucleotide can further comprise a nucleic acid sequence encoding a signal polypeptide operably linked to the 5xe2x80x2 end of the HBsAg encoding sequence. The signal peptide can be selected from the group consisting of a vegetative storage protein (VSP) xcex1S signal peptide and a VSP xcex1L signal peptide.
Additionally, the polynucleotide can comprise an HBsAg encoding sequence further comprising a pre-S region. Optionally, the polynucleotide can comprise an HBsAg encoding sequence which comprises the nucleic acid sequence optimized for expression in plants shown in SEQ ID NO:3. The polynucleotide can be selected from the group of polynucleotides consisting of HB104, HB105, HB106, HB107, HB111, HB114, HB115, HB116, HB117, HB118, HB119, HB120, HB121, HB122, HB123, HB131, HB140.3 HB145 and HB165.
In yet another embodiment of the invention an expression vector comprising a polynucleotide described above is provided. The expression vector can comprise a selectable marker, an E. coli origin of replication, and/or an Agrobacterium tumefaciens origin of replication.
In another embodiment of the invention an E. coli cell transformed with the expression vector described above is provided. A virus like particle can assemble in the cell.
In even another embodiment of the invention an Agrobacteriurn cell is transformed with the expression vector described above. A virus like particle can assemble in the cell. The Agrobacterium cell can further comprise a helper Ti plasmid.
In still another embodiment of the invention a transgenic plant cell comprising the polynucleotide, as described above, is provided. A virus like particle can be assembled in the cell. The plant cell can be selected from the group consisting of tomato, potato, banana, and carrot cells. The plant expression vector can be integrated into the nuclear genome of the plant cell.
In yet another embodiment of the invention a plant seed comprises the plant expression vector as described above.
In still another embodiment of the invention an immunogenic composition comprising any of the plant cells described above is provided. The plant cell can be present in plant tissue selected from the group consisting of a fruit, leaf, tuber, plant organ, seed protoplast, and callus. The immunogenic composition can comprise juice or extract of the plant cell. The immunogenic composition can also comprise an adjuvant. The adjuvant can be expressed as a fusion protein with an antigen of the immunogenic composition.
In yet another embodiment of the invention a method of eliciting an immune response in a mammal is provided. The method comprises the step of administering the composition described above to a human or animal. An immune response is elicited. The composition can be administered orally. The administration can comprise consuming the transgenic plant cell. The polypeptide can be administered by a technique selected from the group consisting of intramuscular, oral, intradermal, intraperitoneal, subcutaneous, and intranasal. Optionally an adjuvant can be administered. The adjuvant can be selected from the group consisting of cholera toxin (CT), E. coli heat labile toxin (LT), anti-idiotypic antibody 2F 10, colonization factor, shiga-like toxin, and intimin. The immune response elicited can be selected from the group of immune responses consisting of humoral; mucosal; cellular; humoral and mucosal; humoral and cellular; mucosal and cellular; and humoral, mucosal and cellular.
In another embodiment of the invention a method of isolating a recombinant HBsAg polypeptide expressed in a plant material is provided. The method comprises subjecting the plant material to a detergent having a concentration of greater than 0.1% and less than 0.5%.
In even another embodiment of the invention a transgenic plant or plant cell is provided. When the transgenic plant or plant cell is consumed as a foodstuff in four or less feedings, it elicits an immune response comprising anti-HBsAg serum antibodies of greater than 50 mIU/mL in a mammal. The immune response can be a primary immune response. The transgenic plant cell can comprise any plant cell described above.
Still another embodiment of the invention provides a transgenic plant or plant cell that, when consumed as a foodstuff in four or less feedings, elicits an anti-HBsAg boosting immune response that increases serum anti-HBsAg antibody levels at least four-fold or to levels greater than 500 mIU/mL in a mammal. The transgenic plant or plant cell can comprise any plant cell described above.
As used herein, an xe2x80x9cantigenxe2x80x9d is a macromolecule capable of eliciting an immune response in a human or in an animal.
An xe2x80x9cepitopexe2x80x9d is a portion of an antigen that comprises the particular part of the antigen to which the antibody binds.
A xe2x80x9ccolonization or virulence antigenxe2x80x9d is an antigen of a pathogenic microorganism that is associated with the ability of the microorganism to colonize or invade its host.
A xe2x80x9cpolynucleotide,xe2x80x9d xe2x80x9cnucleic acid,xe2x80x9d and the like is a polynucleotide that encodes a polypeptide. The polynucleotide or nucleic acid can include introns, marker genes, signal sequences, regulatory elements, such as promoters, enhancers and termination sequences, and the like.
A first polynucleotide is xe2x80x9cnon-identicalxe2x80x9d to a second polynucleotide where at least one nucleotide, and up to and including 85% of overall identity of the first polynucleotide, is different from that of the second polynucleotide.
An xe2x80x9cexpression vectorxe2x80x9d is a plasmid, such as pBR322, pUC, or ColE1; a virus such as an adenovirus, Sindbis virus, simian virus 40, alphavirus vectors, and cytomegalovirus and retroviral vectors, such as murine sarcoma virus, mouse mammary tumor virus, Moloney murine leukemia virus, and Rous sarcoma virus. Bacterial vectors, such as Salmonella ssp., Yersinia enterocolitica, Shigella spp., Vibrio cholerae, Mycobacterium strain BCG, and Listeria monocytogenes can be used. Minichromosomes such as MC and MC1, bacteriophages, virus particles, virus-like particles, cosmids (plasmids into which phage lambda cos sites have been inserted) and replicons (genetic elements that are capable of replication under their own control in a cell) can also be used as expression vectors. Preferably, an expression vector is capable of transforming eukaryotic cells, including, for example plant tissue.
A xe2x80x9cfoodstuffxe2x80x9d or xe2x80x9cedible plant materialxe2x80x9d and the like is any plant material that can be directly ingested by animals or human as a nutritional source or dietary complement. An edible plant material includes a plant or any material obtained from a plant, which is suitable for ingestion by mammal or other animals including humans. This term is intended to include raw plant material that may be fed directly to animals or processed plant material that is fed to animals, including humans.
An xe2x80x9cimmune responsexe2x80x9d comprises the response of a host to an antigen. A humoral immune response comprises the production of antibodies in response to an antigen or antigens. A cellular immune response includes responses such as a helper T-cell (CD4+) response and a cytotoxic T-cell lymphocyte (CD8+) response. A mucosal immune response (or secretory immune response) comprises the production of secretory (sIgA) antibodies. An immune response can comprise one or a combination of these responses.
An xe2x80x9cimmunogenic agentxe2x80x9d or immunogenic polypeptidexe2x80x9d is an antigen or antigens capable of eliciting an immune response. Preferably, the immune response is elicited in a human or animal upon oral ingestion of a eukaryotically expressed antigen. An xe2x80x9cimmunogenic compositionxe2x80x9d contains one or more immunogenic agents, optionally in combination with a carrier, adjuvant, or the like.
A xe2x80x9cfusion proteinxe2x80x9d is a protein containing at least 2, 3, 4, 5, 10, or more same or different amino acid sequences linked in a polypeptide where the sequences are not natively expressed as a single protein. Fusion proteins can be produced by well known genetic engineering techniques.